Bottom Line:
Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity.Apoptotic responses to a variety of stimuli were not affected.These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

ABSTRACTThe dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

Figure 5: MKK7 regulates BMMC proliferation but not apoptosis. (A and B) Proliferation of mkk7+/+ and mkk7−/− BMMCs. mkk7+/+ and mkk7−/− BMMCs were incubated with increasing concentrations of (A) IL-3 or (B) SCF (c-Kit-Ligand). [3H]Thymidine uptake was determined 24 h after cytokine addition. Similar results were obtained for five separate cultures from five mice of each genotype. (C) Induction of apoptosis in mkk7+/+ and mkk7−/− BMMCs. Cells were stimulated for 14 or 42 h with either anisomycin (Aniso; 10 μM), UV-irradiation (500 mJ), or heat shock (45°C for 30 min). Cell viability was determined in triplicate by 7-AAD and PI staining and normalized to the percentage of viable cells in untreated cultures. One result of a triplicate culture (± SD) representative of five independent experiments is shown for each activation. Normal susceptibility to cell death of mkk7−/− BMMCs (P > 0.1) was also observed at various seeding numbers and in response to growth factor deprivation and osmotic shock (not shown).

Mentions:
SAPKs/JNKs and their activators MKK4 and MKK7 are expressed in all hematopoietic lineages. However, the role of MKK7 and SAPKs/JNKs in the function of non-lymphoid hematopoietic cells has not been explored using mutational analysis. Our targeting strategy of mutating one mkk7 allele with a Neo-cassette and the other allele with a hygromycin-resistance vector allowed us to study the role of MKK7 in a non-lymphoid hematopoietic lineage, BMMC lines. Because mast cells derived from mkk7−/− ES cells in the chimeric mice (but not those derived from rag1−/− ES cells) were resistant to hygromycin, several BMMC lines known to be mkk7−/− could readily be established (Fig. 1 E). As controls, we generated mkk7+/+ BMMCs from 129/Ola mice, which have the same genetic background as the mkk7−/− E14 ES cells. BMMCs of both genotypes showed similar expression levels of c-Kit (SCF-R) and the IgE receptor (not shown), two markers characteristic of mature BMMCs 37. Thus, loss of MKK7 expression does not prevent the emergence and differentiation of BMMCs. However, the proliferation of mkk7−/− BMMCs in response to the mast cell growth factor IL-3 (Fig. 5 A) or SCF (c-Kit-ligand; Fig. 5 B) was strikingly increased compared with that of wild-type BMMCs, paralleling the hyperproliferation observed in mkk7-deficient thymocytes and B cells. Again, no significant differences between mkk7+/+ and mkk7−/− BMMCs were observed in apoptosis induced by growth factor deprivation or osmotic stress (NaCl) (not shown), or by anisomycin, heat shock, or UV-irradiation (Fig. 5 C). Similar results were obtained using in vitro differentiated FcεR+c-Kit+ mast cell lines from mkk7+/+, mkk7+/−, and mkk7−/− ES cells that were selected and differentiated under identical culture conditions (not shown) 28. These data indicate that loss of MKK7 results in the hyperproliferation of both lymphoid and non-lymphoid hematopoietic cells in response to multiple growth factor or antigen receptor stimulation.

Figure 5: MKK7 regulates BMMC proliferation but not apoptosis. (A and B) Proliferation of mkk7+/+ and mkk7−/− BMMCs. mkk7+/+ and mkk7−/− BMMCs were incubated with increasing concentrations of (A) IL-3 or (B) SCF (c-Kit-Ligand). [3H]Thymidine uptake was determined 24 h after cytokine addition. Similar results were obtained for five separate cultures from five mice of each genotype. (C) Induction of apoptosis in mkk7+/+ and mkk7−/− BMMCs. Cells were stimulated for 14 or 42 h with either anisomycin (Aniso; 10 μM), UV-irradiation (500 mJ), or heat shock (45°C for 30 min). Cell viability was determined in triplicate by 7-AAD and PI staining and normalized to the percentage of viable cells in untreated cultures. One result of a triplicate culture (± SD) representative of five independent experiments is shown for each activation. Normal susceptibility to cell death of mkk7−/− BMMCs (P > 0.1) was also observed at various seeding numbers and in response to growth factor deprivation and osmotic shock (not shown).

Mentions:
SAPKs/JNKs and their activators MKK4 and MKK7 are expressed in all hematopoietic lineages. However, the role of MKK7 and SAPKs/JNKs in the function of non-lymphoid hematopoietic cells has not been explored using mutational analysis. Our targeting strategy of mutating one mkk7 allele with a Neo-cassette and the other allele with a hygromycin-resistance vector allowed us to study the role of MKK7 in a non-lymphoid hematopoietic lineage, BMMC lines. Because mast cells derived from mkk7−/− ES cells in the chimeric mice (but not those derived from rag1−/− ES cells) were resistant to hygromycin, several BMMC lines known to be mkk7−/− could readily be established (Fig. 1 E). As controls, we generated mkk7+/+ BMMCs from 129/Ola mice, which have the same genetic background as the mkk7−/− E14 ES cells. BMMCs of both genotypes showed similar expression levels of c-Kit (SCF-R) and the IgE receptor (not shown), two markers characteristic of mature BMMCs 37. Thus, loss of MKK7 expression does not prevent the emergence and differentiation of BMMCs. However, the proliferation of mkk7−/− BMMCs in response to the mast cell growth factor IL-3 (Fig. 5 A) or SCF (c-Kit-ligand; Fig. 5 B) was strikingly increased compared with that of wild-type BMMCs, paralleling the hyperproliferation observed in mkk7-deficient thymocytes and B cells. Again, no significant differences between mkk7+/+ and mkk7−/− BMMCs were observed in apoptosis induced by growth factor deprivation or osmotic stress (NaCl) (not shown), or by anisomycin, heat shock, or UV-irradiation (Fig. 5 C). Similar results were obtained using in vitro differentiated FcεR+c-Kit+ mast cell lines from mkk7+/+, mkk7+/−, and mkk7−/− ES cells that were selected and differentiated under identical culture conditions (not shown) 28. These data indicate that loss of MKK7 results in the hyperproliferation of both lymphoid and non-lymphoid hematopoietic cells in response to multiple growth factor or antigen receptor stimulation.

Bottom Line:
Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity.Apoptotic responses to a variety of stimuli were not affected.These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

ABSTRACTThe dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.